No abstract

Prevention of multidrug-resistant (MDR) bacterial infections relies on accurate detection of these organisms. We investigated shotgun metagenome sequencing for the detection of methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus (VRE), and MDR Enterobacteriaceae. Fecal metagenomes were analyzed from high-risk inpatients and compared to those of low-risk outpatients and controls with minimal risk for a MDR bacterial infection. Principal-component analysis clustered patient samples into distinct cohorts, confirming that the microbiome composition was significantly different between cohorts (P ‫؍‬ 0.006). Microbial diversity and relative anaerobe abundance were preserved in outpatients compared to those in controls. Relative anaerobe abundance was significantly reduced in inpatients compared to that in outpatients (P ‫؍‬ 0.006). Although the potential for MDR bacteria was increased in inpatients and outpatients compared to that in controls (P < 0.001), there was no difference between inpatients and outpatients. However, 9 (53%) inpatients had colonization with a MDR bacterium that was not identified by culture. Unlike culture, shotgun sequencing quantitatively characterizes the burdens of multiple MDR bacteria relative to all of the microbiota within the intestinal community. We propose consideration of key microbiome features, such as diversity and relative anaerobe abundance, in addition to the detection of MDR bacteria by shotgun metagenome sequencing as a novel method that might better identify patients who are at increased risk of a MDR infection. Multidrug-resistant (MDR) bacterial infections cause significant morbidity and mortality and are increasingly common in children (1-3). Clinically important MDR bacteria include methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus (VRE), and MDR Enterobacteriaceae with AmpC beta-lactamase resistance (AmpC), extended-spectrum beta-lactamase (ESBL) resistance, or carbapenem-resistant betalactamases (CRE). Antimicrobial exposures reduce the commensal gut anaerobes with compensatory increased Enterobacteriaceae and Enterococcus abundances as well as bacteriophages, allowing transmission of antibiotic resistance genes between species (4). This results in the loss of host resistance to abnormal fecal colonization and may select for MDR bacteria (5-7). Subsequently, the intestines serve as the primary reservoir of MDR bacteria (8, 9). Increased abundances of these organisms within the intestines precede the onset of an invasive bloodstream infection (BSI), and the risk of infection is related to the density of colonization (10-13).Timely and accurate detection of MDR bacteria is crucial to prevent the spread of MDR bacteria and to decrease rates of MDR infections (14). There is an urgent need for the development of new diagnostic methods, including metagenomics, to combat rising antibiotic resistance rates, which was explicitly stated in recent national action plans by the CDC and the White House (15, 16). D...

Clostridium difficile impairs Paneth cells, driving intestinal inflammation that exaggerates colitis. Besides secreting bactericidal products to restrain C. difficile, Paneth cells act as guardians that constitute a niche for intestinal epithelial stem cell (IESC) regeneration. However, how IESCs are sustained to specify Paneth-like cells as their niche remains unclear. Cytokine-JAK-STATs are required for IESC regeneration. We investigated how constitutive STAT5 activation (Ca-pYSTAT5) restricts IESC differentiation towards niche cells to restrain C. difficile infection. We generated inducible transgenic mice and organoids to determine the effects of Ca-pYSTAT5-induced IESC lineages on C. difficile colitis. We found that STAT5 absence reduced Paneth cells and predisposed mice to C. difficile ileocolitis. In contrast, Ca-pYSTAT5 enhanced Paneth cell lineage tracing and restricted Lgr5 IESC differentiation towards pYSTAT5+Lgr5−CD24+Lyso+ or cKit+ niche cells, which imprinted Lgr5hiKi67+ IESCs. Mechanistically, pYSTAT5 activated Wnt/β-catenin signaling to determine Paneth cell fate. In conclusion, Ca-pYSTAT5 gradients control niche differentiation. Lack of pYSTAT5 reduces the niche cells to sustain IESC regeneration and induces C. difficile ileocolitis. STAT5 may be a transcription factor that regulates Paneth cells to maintain niche regeneration.

Under conditions of altered microbial ecology, C. difficile incites epithelial injury and marked intestinal inflammation, the primary determinant of disease outcome. Restoration of a diverse intestinal microbial population by fecal microbiota transplantation attenuates disease and prevents recurrence by mechanisms that are yet to be fully elucidated.

Mechanisms underlying susceptibility to anthrax infection are unknown. Using a phylogenetically diverse panel of inbred mice and spores of Bacillus anthracis Ames, we investigated host susceptibility to pulmonary anthrax. Susceptibility profiles for survival time and organ pathogen load differed across strains, indicating distinct genetic controls. Tissue infection kinetics analysis showed greater systemic dissemination in susceptible DBA/2J (D) mice but a higher terminal bacterial load in resistant BALB/cJ (C) mice. Interestingly, the most resistant strains, C and C57BL/6J (B), demonstrated a sex bias for susceptibility. For example, BALB/cJ females had a significantly higher survival time and required 4-fold more spores for 100% mortality compared to BALB/cJ males. To identify genetic regions associated with differential susceptibility, survival time and extent of organ infection were assessed using mice derived from two susceptibility models: (i) BXD advanced recombinant inbred strains and (ii) F2 offspring generated from polar responding C and D strains. Genomewide analysis of BXD strain survival identified linkage on chromosomes 5, 6, 9, 11, and 14. Quantitative trait locus (QTL) analysis of the C؋DF2 population revealed a significant QTL (designated Rpai1 for resistance to pulmonary anthrax infection, locus 1) for survival time on chromosome 17 and also identified a chromosome 11 locus for lung pathogen burden. The striking difference between genome-wide linkage profiles for these two mouse models of anthrax susceptibility supports our hypothesis that these are multigenic traits. Our data provide the first evidence for a differential sex response to anthrax resistance and further highlight the unlikelihood of a single common genetic contribution for this response across strains.Anthrax is one of the most ancient and lethal human diseases caused by the virulent (toxigenic and encapsulated) strains of Bacillus anthracis. In humans, anthrax may take three forms depending upon the route of infection, namely, pulmonary (inhalational), cutaneous, and gastrointestinal, with pulmonary being the most lethal and difficult to treat. While the naturally acquired pulmonary anthrax is relatively rare (occurring only ϳ5% as often as cutaneous anthrax), this form has a high potential for misuse as a weapon of bioterrorism, considering that environmental dissemination is the most expected mode of release of the agent in mass attacks (26). Pulmonary anthrax often proves fatal, with mortality approaching 100% if not treated early. Anthrax spores have long been considered a potential agent of biological warfare (25, 50, 58). Prior to the 11 cases of pulmonary anthrax that occurred via the U.S. mail delivery system in 2001 (29), fatal cases of pulmonary anthrax in the United States and other countries have been associated with occupational and accidental exposures (37,60). But the bioterrorism attack in 2001 exposed a new cause for serious concern for future and more widespread attacks on military and civilian populations.The ...